Topical antibacterial preparation

Abstract: The present invention relates to a topical antibacterial preparation for the treatment of infected hands and for the advancement of their healing. Said preparation comprises silver sulfadiazine (SSD) and collagen, in which the ratio of W/W of collagen to SSD ranges from 4 parts collagen to 1 part SSD, to 200 parts collagen to 1 part SSD. The concentration of the SSD is preferably 0.5% to 2.5%, advantageously 1%. The collagen is advantageously native collagen, which may be acid soluble or insoluble, or mixtures thereof. The preparation may comprise an inert excipient, e.g. Si0₂, or blood plasma components, peptides, carbohydrates.

Description: The present invention relates to a topical antibacterial preparation for the treatment of infected wounds and for the enhancement of their healing. Silver sulfadiazine (SSD) preparations are among the best topical antibacterial agents in clinical use for the treatment of infected wounds, especially of burns. However, because SSD is water-insoluble, its most commonly known preparations consist of dispersions with water-soluble cream bases containing, inter alia, emulsifying substances that not only do not contribute to the healing process but frequently even hinder it. Moreover, said known preparation cannot be used in a dry form. Finally the amount of SSD required in said emulsions as suggested by various manufacturers is about 318 - 530 mg in an area of 10 x 10 cm in a 3-5 mm layer. These large amounts of SSD may cause systemic absorption and damage to the wound tissue. From EP-A-140 596 there is known a porous complex of a biodegradable fibrous protein, e.g. collagen, with a polyanionic plant, polysaccharide. S.S.D. may be incorporated into the wound contact layer. It should be stressed that there is concerned a complex and not a simple mixture and/or compound which requires the presence of a special polysaccharide. Said polysaccharide gives special properties to the complex which are described and claimed. Collagen is the major connective tissue structural protein with a variety of biological properties that are intimately involved in all the phases of tissue repair processes following injury. The collagen molecule has several hydrophobic domains along its long polypeptide chains which are suitable for hydrophobic interactions with other hydrophobic molecules, such as silver sulfadiazine. It has been found that by interacting collagen with SSD one has no need to use emulsifying agents, thus obtaining an active antibacterial preparation which also enhances the healing process at the same time. From US-A-4 581 028 is known an infection-resistant device which is a vascular graft prosthesis for use within the interior of a human or animal body which may comprise collagen and SSD. However, said device cannot be used as a topical preparation. However, the amount of SSD utilised cannot be calculated. From US-A-4 599 226 is known a method for preparing animal tissues for use as burn or wound dressings comprising, inter alia, SSD. It is assumed that collagen may be present in the skin tissue utilised. However, nothing can be learned about a topical preparation comprising collagen and SSD. From DE-A-3 523 023 A1 is known a collagen sponge comprising 0.5-10 mg of a silver salt which dissolves with difficulty. However, only the use of inorganic silver salts is shown and a very specific method of preparation is exemplified. EP-A-480 189 discloses a composition for the treatment of sores, ulcerations and burns containing 0.1 - 0.5% by weight of hyaluronic acid sodium salt, 1% by weight of SSD, 2-7% by weight of glycerin, 4% by weight of non-denatured collagen, 1-2% by weight of sorbitol and distilled water up to 100%. Thus, the sodium salt of hyaluronic acid is an essential component of the composition whereas SSD is only one of several possible disinfectants. It has thus been desirable to find a specific topical preparation which overcomes the above disadvantages, is easy to prepare and has the required amounts of SSD. The present invention provides a composition comprising silver sulfadiazine (SSD) and collagen, wherein the weight ratio of collagen to SSD ranges from 4 parts collagen to 1 part SSD to 200 parts collagen to 1 part SSD and wherein the minimum concentration of SSD is 0.5%; a composition consisting of 0.1 - 0.5% by weight of hyaluronic acid sodium salt, 1% by weight of SSD, 2-7% by weight of glycerin, 4% by weight of non-denatured collagen, 1-2% by weight of sorbitol and distilled water up to 100% by weight being excluded. Specific embodiments of said composition are defined in the dependent claims. In a preferred embodiment of the invention said composition comprises an inert excipient, e.g. colloidal silicon dioxide (SiO 2 ). The collagen indicated herein is preferably native collagen of type I, which may be either pure or comprise other types of collagen, e.g. type III and in which the triple helical conformation of the molecule is preserved. The native collagen may be acid soluble (ASC) or insoluble (ISC) or a combination of both. The ACS can be prepared from any source that is rich in type I, such as, tendons, bovine or goat hide or human placenta. A preferred source is bovine hide. Suitable methods for the preparation of ASC are described in The Methodology of Connective Tissue Research, edited by David A. Hall, Chapter 3, - Preparation of Acid and Citrate Soluble Collagen. A specific method is described by J. Gross (J. Exp. Med. 107: 247-263, 1958), but as indicated above, other methods are also suitable. There are known various methods to prepare ISC. Suitable methods are described in the above book edited by David A. Hall, chapter 1, Z. Deyl and M. Adam, pages 1-8. A suitable method is described in EP-A-301 977. However, there may also be indicated further ones, such as a collagen crosslinked with starch dialdehyde. Specific methods are described also in (1) Bairati, A. et al. - J.Submicr. Cytol 4: 89, 1972; (2) Gupta, R.L. et al. - Indian J. Surg. 646, 1978; (3) Woodroof, E.A. - J.Bioeng. 2: 1, 1078; and (4) Chvapil, M. et al. - J.Surg.Res. 35: 402, 1983. The composition according to the present invention may be used in the dry form or in a liquid form. It may comprise additional materials, eg. blood plasma components, peptides and carbohydrates. The composition according to the present invention is suitably prepared by adding, with constant stirring, the required amount of SSD admixed with SiO 2 , if present, to an appropriate cold (0-4°C) solution of the collagen in 0.1 - 0.5M acetic acid. This is then followed by mild vortexing the composition obtained for about 10-15 minutes, after which it is lyophilized, sterilized and stored under suitable conditions. The sterilization is performed preferably by gamma irradiation. The composition according to the present invention may also be prepared in the form suitable for reconstitution as a liquid preparation. After preparation as described above, the lyophilized material is dissolved in 0.1 - 0.5M acetic acid and then dialyzed against a suitable buffer (e.g. Na or K phosphate buffer, TRIS buffer) adjusted to pH 7.4 - 7.6. The preparation is lyophilized, suitably packed and sterilized. It is then ready for reconstitution by the addition of an appropriate quantity of sterile purified water. The present invention will now be illustrated with reference to the following Examples without being limited by them. In all Examples 1-3, collagen stands for native collagen. Example 1 The following composition was prepared: SSD 1% Colloidal silicon dioxide (Aerosil ® 300 Degussa, Germany) 0.05% Collagen to 100% The composition was prepared as follows: The SSD was mixed with the Aerosil® and the mixture was dispersed in a 0.5 M acetic acid solution of collagen. The preparation was subsequently dried by lyophilization. a. (ASP) The preparation (ASP) consisted of a powder in which mainly acid soluble type I collagen was used. b. (ASS) The preparation (ASS) consisted of a sponge in which mainly acid soluble type I collagen was used. Said sponge was weighed and measured. The specific gravity was approximately 0.0107 g/cc. Such a sponge of 0.3 cm thickness and 10 x 10 cm area contains about 3.2 mg of SSD. c. (ISP) The preparation (ISP) consisted of a powder in which mainly insoluble type I collagen was used. d. (ISS) The preparation (ISS) consisted of a sponge in which mainly insoluble type I collagen was used. Results of tests performed with Examples a-d A) In vitro tests were performed to determine the bacteriostatic and bacteriocidal properties of the preparations. The object of this study was to test whether the SSD in said preparations would still show its antibacterial activity in vitro. Samples of each of the four preparations were placed as isolated foci on Blood-Agar plates which were seeded with either Pseudomonas aeruginosa, Escherichia coli or Staphylococcus aureus. The growth of the microorganisms was monitored after incubation for 24 and 48 hours. Bacterial growth was inhibited around all the samples containing SSD. The tests were repeated in several different variations, all with the same results, indicating an effective inhibition of bacterial growth (bacteriostasis) or an effective control of a contaminated field (bacteriocidal activity).All the samples containing collagen with SSD were thus found to be equally effective as both infection-preventing and infection-combating agents in vitro. B) Preliminary in vivo studies were performed to test the effect of the different preparations on the healing process of non-infected dermal wounds. The object of this study was to ascertain that the various preparations, when placed on wounds, did not impair the healing process. Full-thickness dermal excision wounds were inflicted on the back of anaesthetized guinea pigs, which were then treated with either of said preparations, or left untreated. An equal amount of either preparation was placed on the wound. The results were assessed histologically after 10 days. They showed that none of the preparations had a deleterious effect on the healing process. Moreover, healing was even enhanced with the preparations containing SSD as compared to those left untreated. The rate of closure was as follows: Control (untreated) 50% closure ASS 60% closure ISS 80% closure ISP 80% closure ASP 100% closure C) Preliminary in vivo studies were performed on the effect of the different preparations on the bacterial count in infected wounds. The object of this study was to test the ability of the preparations to reduce the bacterial count in infected wounds in vivo. Full-thickness dermal excision wounds were inflicted on the back of anaesthetized guinea pigs and infected with about 1 X 10 6 bacteria. After 30 minutes the wounds were covered with either of said preparations, or left untreated. Bacterial counts were performed on day 1, day 3, day 7 and day 10. At each time point, the wound samples were treated with a Stomacher® Lab Blender and the number of viable bacteria in the wound were counted by the agar-plate method. In all the samples from wounds treated with preparations containing SSD there was a drastic and significant drop in the bacterial count lasting 7 to 10 days, as compared with the untreated contaminated wounds. Example 2 The following composition was prepared: Silver Sulfadiazine 5% Aerosil® 300 0.25% Collagen to 100% The silver sulfadiazine was mixed with the Aerosil® and the mixture was dispersed in a 0.5 M acetic acid solution of collagen. The preparation was subsequently lyophilized. In vitro tests to determine the antibacterial activity were carried out. Samples of the preparation were placed as isolated foci on Mueller Hinton agar plates which were seeded with either Escherichia coli or Staphylococcus aureus. The growth of the microorganisms was monitored for 24 and 48 hours. Bacterial growth was inhibited around all the samples. Example 3 The following composition was prepared: Silver Sulfadiazine 20% Aerosil® 300 1% Collagen to 100% The silver sulfadiazine was mixed with Aerosil® and the mixture was dispersed in a 0.5 M acetic acid solution of collagen. The preparation was subsequently lyophilized. In vitro tests to determine the antibacterial activity were carried out. Samples of the preparation were placed as isolated foci on Mueller Hinton agar plates which were seeded with either Escherichia coli or Staphylococcus aureus. The growth of the microorganisms was monitored for 24 and 48 hours. Bacterial growth was inhibited around all the samples.

Claims: A composition comprising silver sulfadiazine (SSD) and collagen, wherein the weight ratio of collagen to SSD ranges from 4 parts collagen to 1 part SSD to 200 parts collagen to 1 part SSD and wherein the minimum concentration of SSD is 0.5%; a composition consisting of 0.1 - 0.5% by weight of hyaluronic acid sodium salt, 1% by weight of SSD, 2-7% by weight of glycerin, 4% by weight of non-denatured collagen, 1-2% by weight of sorbitol and distilled water up to 100% by weight being excluded. A composition according to claim 1, wherein the concentration of SSD is from 0.5% to 2.5%. A composition according to claim 1, wherein the concentration of SSD is 1%. A composition according to any one of claims 1-3, wherein the collagen is mainly native collagen. A composition according to claim 4, wherein the native collagen is acid soluble collagen (ASC). A composition according to claim 4, wherein the native collagen is insoluble collagen (ISC). A composition according to claim 4, wherein the native collagen is a mixture of ASC and ISC. A composition according to any one of claims 1-7, additionally comprising colloidal silicon dioxide.

Inventors: MARCOS DAVID, SHOSHAN SHMUEL, MARCOS, DAVID, SHOSHAN, SHMUEL
Applicants: YISSUM RES DEV CO, YISSUM RESEARCH DEVELOPMENT COMPANY OF THE HEBREW UNIVERSITY OF JERUSALEM
Application year: 1994
Main classification: A61L15
IPC codes: A61K47, A61K38, A61K31, A61L26, A61P17, A61K9